Abstract

Decreased oxygen delivery to cells (hypoxia) is prevalent in a number of important diseases. Little is known about mechanisms of oxygen sensing at the cellular level or about whether functional correlates of oxygen sensing exist. In this study, we examined the impact of hypoxia on stimulated epithelial ion transport function. T84 cells, a model of intestinal epithelia, were grown on permeable supports, exposed to hypoxia (range 1–21% O2) for periods of time between 0 and 72 h and assessed for stimulated ion transport. Hypoxia evoked a specific decrease in cyclic nucleotide-stimulated (cAMP and cGMP) but not Ca++-stimulated ion transport. 86Rb (K+ tracer) uptake and 125I (Cl−tracer) efflux were reduced in hypoxic cells by >50% and >40%, respectively, fluid movement was reduced by hypoxia (>50% decrease) and reoxygenation resulted in partial recovery of the ion transport responses. Stimulated and basal levels of both cAMP and cGMP were decreased in response to hypoxia, although intracellular ATP levels were unaltered under similar conditions. Exogenous addition of cobalt, nickel or manganese, all of which compete for oxygen binding on heme-containing proteins, mimicked hypoxia. Because guanylate cyclase is a heme protein, we measured the influence of cobalt on activity of guanylate cyclase in purified plasma membrane preparations and found cobalt to inhibit stimulated cGMP levels in this cell-free system. Finally, pharmacological lowering of intracellular cGMP (using LY83583) resulted in decreased cAMP-stimulated Cl− secretion, and direct elevation of cGMP (using 8-bromo-cGMP or dibutyryl-cGMP) restored this hypoxia-induced activity. We conclude that a potential oxygen-sensing mechanism of epithelial cells involves the cooperation of heme-containing proteins such as guanylate cyclase and that biochemical cross-talk between cAMP- and cGMP-stimulated pathways may be important in such responses.

Cells of the body are commonly exposed to decreased levels of oxygen, a condition termed hypoxia. The ability of cells to tolerate and adapt to acute, and sometimes severe, hypoxia is crucial to survival. Surprisingly little is known about the basic oxygen-sensing mechanisms and adaptive strategies invoked during cellular hypoxia. Significant evidence indicates that cellular adaptation to hypoxia varies greatly across species boundaries and among cell types (Hochachka, 1986). Interestingly, most mammalian cells have limited ability to cope with oxygen deprivation and consequently are easily damaged by hypoxia (Stevens and Rodman, 1995).

Tissue hypoxia is commonly associated with a number of important diseases. Recent evidence from a number of laboratories indicates that the damaging effects of cellular hypoxia are likely not mediated by direct oxygen deprivation (Waxman, 1996). Rather, a new paradigm has evolved to indicate that hypoxia may “prime” cellular machinery for damage mediated by additional physiological stimuli (Waxman, 1996). Such physiological stimuli may include inflammatory cytokines (Clarket al., 1995; Colgan et al., 1996; Shreeniwaset al., 1992), bioactive lipids (Michiels et al., 1993), bacterial toxins (Waxman, 1996; Zünd et al., 1996b) and reactive oxygen intermediates (Mertens et al., 1990). This universal “priming” effect of diverse signals suggests that responses elicited by hypoxia involve a basic cellular event common to a number of signal transduction pathways.

A potential mechanism of adaptation to hypoxia at the cellular level could involve the functional regulation of nucleotide cyclases, cellular enzyme systems that catalyze the conversion of intracellular ATP/GTP to cyclic AMP/GMP (Taussig and Gilman, 1995). Indeed, we (Zünd et al., 1996b) and others (Ogawa et al., 1992; Stevens and Rodman, 1995; Tretyakov and Farber, 1995) have shown that hypoxia directly regulates intracellular levels of adenine nucleotides in a number of cell types. Moreover, our data indicated that pharmacological regulation of adenylyl cyclase effectively reversed hypoxia-elicited cellular responses (Zündet al., 1996b), and others have shown that preservation of cAMP/cGMP pathways are protective at the whole-organ level (Pinskyet al., 1994; Pinsky et al., 1993). The mechanism(s) by which hypoxia regulates cyclic nucleotide levels is at present poorly understood. Moreover, the generation of intracellular cyclic nucleotides, especially cGMP, involves heme-containing proteins (Stone and Marletta, 1995). Previous investigations by others have shown that molecular oxygen binding to hemoproteins may serve as a mechanism of sensing extracellular oxygen concentrations and, as such, could serve as a signal transduction pathway leading to gene activation (Goldberg et al., 1988). Moreover, it has been shown that extracellular cobalt and nickel can mimic hypoxia by binding within the porphyrin ring of heme and substituting for iron, thus locking heme in a deoxy state (Goldberg et al., 1988). Whether adenylyl or guanylyl cyclase serves to “sense” extracellular O2levels remains to be determined.

Here we examine the impact of hypoxia on functional aspects of cultured intestinal epithelial cells. Our results indicate that epithelial hypoxia specifically down-regulates stimulated electrogenic chloride secretion, the primary transport event responsible for mucosal hydration. Such hypoxia-induced alterations were specific for cyclic nucleotide agonists, were evident at the level of membrane channels/transporters and could be mimicked by exposing epithelia to cobalt. Moreover, both cobalt and hypoxia significantly diminished GC activity and could be partially reversed by the addition of exogenous cGMP. These data indicate a role for heme proteins, such as GC, in epithelial oxygen “sensing” and reveal significant cAMP/cGMP cross-talk during hypoxia.

Materials and Methods

Cell culture.

T84 intestinal epithelial cells (passages 67–85) were grown and maintained as confluent monolayers on collagen-coated permeable supports as previously described in detail (Dharmsathaphorn and Madara, 1990). Monolayers were grown on 0.33-cm2 ring-supported polycarbonate filters (Costar Corp., Cambridge, MA) unless otherwise noted, and they were used 6 to 12 days after plating as described previously (Madara et al., 1992a).

Epithelial cultures were exposed to hypoxia as described previously (Colgan et al., 1996). Growth media were replaced with fresh media equilibrated with hypoxic gas mixture, and cells were placed in the hypoxic chamber (Coy Laboratory Products, Ann Arbor, MI). Oxygen concentrations were as indicated (normoxia equal to 21% O2), the balance being made up of nitrogen, carbon dioxide (constant 5% CO2) and water vapor from the humidified chamber. Monolayers were monitored electrically in hypoxia by interfacing the voltage clamp from the outside through an airtight seal in the chamber.

Electrophysiological measurements.

To measure agonist-stimulated SSC, transepithelial potentials and resistance, we used a commercially available voltage clamp (Iowa Dual Voltage Clamps, Bioengineering, University of Iowa) interfaced with an equilibrated pair of calomel electrodes and a pair of Ag-AgCl electrodes, as described in detail elsewhere (Dharmsathaphorn and Madara, 1990). Cl− secretory responses are expressed as a change in SSC (peak SSC minus base-line SSC; designated ΔSSC) necessary to maintain zero potential difference across the monolayer.

Isotope efflux and uptake assays.

Cl− channel and K+ channel activity were monitored using125I and 86Rb efflux, respectively, on T84 cells grown on 1-cm2 permeable supports, as described before (Colgan et al., 1994; Venglarik et al., 1990). Rate constants of efflux were calculated as [ln(R2) − ln(R1)]/(t2 −t1), where Rx is the percent of radioactivity remaining monolayer-associated at timetx, as reported elsewhere (Venglarik et al., 1990). Bumetanide-sensitive and bumetanide-insensitive components of 86Rb uptake were used to determine Na+/K+/2Cl− cotransporter and Na+-K+-ATPase activity, respectively, as described elsewhere (Matthews et al., 1992). Results of86Rb uptake were corrected for the specific activity of K+ as described previously (Owen and Prastein, 1985).

Fluid transport assay.

The methods for measuring transmonolayer fluid movement were adapted from those described by Smith and Welsh (Smith and Welsh, 1993) and as reported elsewhere (Zünd et al., 1996a). Cells were incubated in hypoxia or normoxia as described above for 24 h. In subsets of monolayers, the cAMP agonists forskolin (50 μM) and IBMX (100 μM) were added to the basolateral solution to promote fluid movement. The apical solution was collected and spun at high speed in an Eppendorf centrifuge, and the recovered fluid was weighed on a balance to determine volume.

Measurement of intracellular ATP.

Confluent T84 monolayers on six well plates were exposed to the indicated experimental conditions. Controls were similarly treated cells exposed to normoxia. After incubation, ATP was extracted from washed monolayers with ice-cold extraction buffer [2% trichloracetic acid and 2 mM EDTA], and lysates were sonicated and cleared by centrifugation at 10,000 × g for 5 min. ATP concentrations were determined from supernatants using a luciferin/luciferase-based assay and a chemiluminometer (Chrono-log Corp., Havertown, PA) as previously described (Colgan et al., 1991).

Measurement of cAMP/cGMP.

Confluent T84 monolayers on six well plates were exposed to the indicated experimental conditions and washed. After incubation, cells were cooled to 4°C, and nucleotides were extracted from washed monolayers with extraction buffer [66% EtOH, 33% HBSS containing the phosphodiesterase inhibitor IBMX, 5 mM (Sigma Chemical Co., St. Louis, MO)]. Lysates were then cleared by spinning at 10,000 × g for 5 min and dried under vacuum to remove EtOH. Samples were rehydrated in water, and cAMP or cGMP was quantified using displacement ELISAs (both from Amersham, Arlington Heights, IL) according to the manufacturer’s instructions. Nucleotide levels were expressed as picograms of cGMP/cAMP per microgram of total protein.

Assay of plasma membrane GC activity.

Plasma membranes for assay of GC activity were prepared as described previously (Parkoset al., 1996), with modifications. Briefly, T84 cells grown to confluence on six well plates were cooled to 4°C, washed with HBSS and scraped from the surface with a Teflon spatula into homogenization buffer consisting of 0.34 M sucrose, 10 mM HEPES, 1 mM ATP, 0.1 M EDTA, 1 mM dithiothreitol and protease inhibitors (chymostatin, aprotinin and PMSF). Scraped cells were homogenized with a dounce homogenizer at 4°C, nuclear debris was removed by centrifugation at 1000 ×g and NaCl concentration was adjusted to 1 M to remove peripheral membrane proteins. The resulting membrane suspension was pelleted by ultracentrifugation at 100,000 × g for 45 min, and the membrane pellet was resuspended in phosphate buffered saline. Protein concentrations were determined using the Bradford assay (Bradford, 1976), and 50 μg total protein was added to cGMP assay buffer [1 mM EDTA, 5 mM MgSO4, 3 U/ml creatine kinase, 5 mM creatine phosphate, 5 mM IBMX, 1.5 mM GTP, pH 7.4] in the presence or absence of heat-stable enterotoxin from E. coli (STa, 100 ng/ml) and in the presence or absence of CoCl2 (333 μM) or FeCl2 (333 μM) for 15 min at 37°C. The reaction was terminated by incubation at 90°C for 10 min, samples were centrifuged at 14,000 × g for 5 min and cGMP from supernatants was measured as described above.

Pharmacological alterations of intracellular cGMP in epithelia.

In subsets of experiments, intracellular cGMP levels were specifically diminished using 6-anilino-5,8-quinolinedione (LY83583, Biomol Inc., Plymouth Meeting, PA), which decreases intracellular cGMP but not cAMP levels (Schmidt et al., 1985), or elevated using 8-bromo-cGMP or dibutyryl-cGMP (Sigma). In both cases, normoxic or hypoxic epithelial monolayers were preexposed to agent for 30 min at 37°C, washed into HBSS containing equivalent concentrations of agent and assessed for forskolin (1 μM)-stimulated Cl secretion, as described above.

Data presentation.

Electrophysiological fluid transport and cGMP data were compared by two-factor ANOVA, Student’s ttest or Wilcoxon’s signed rank test, where appropriate. Values are expressed as the mean and S.E.M. of n monolayers from at least three separate experiments.

Epithelial exposure to hypoxia attenuates forskolin-stimulated SSC and fluid transport responses. T84 intestinal epithelia were exposed to normoxia (21% O2) or hypoxia (1% O2) for the indicated periods of time (panel A) or at the indicated concentrations of O2 (panel B) and examined for forskolin (10 μM)-stimulated Cl− secretion (measured as a SSC). Hypoxia did not significantly influence epithelial barrier function (see panel A inset). Data are pooled from nine individual monolayers from three experiments in each condition, and results are expressed as the mean ± S.E.M. ΔSSC. Panel C demonstrates epithelial fluid transport across monolayers exposed to normoxia (solid bars, 21% O2, 24 h) or hypoxia (hatched bars, 1% O2, 24 h) in the absence (media only) or presence of cAMP stimulation (50 μM forskolin and the phosphodiesterase inhibitor IBMX at 50 μM). Values represent the recovered fluid volume after 24 h. Data are pooled from 5 to 7 monolayers each, and results are expressed as the mean ± S.E.M.

Epithelial electrogenic Cl− secretion can be stimulated by multiple agonists mediated by elevations in cAMP, cGMP or Ca++ (Barrett, 1993). As shown in table1, we used a panel of Cl−secretagogues acting through different mechanisms to determine agonist specificity for hypoxia-elicited down-regulation of Cl−secretion. Epithelial exposure to hypoxia significantly diminished both cAMP-stimulated (forskolin, 8-bromo-cAMP, adenosine, 5′ AMP, VIP) and cGMP-stimulated (STa) Cl− secretion but did not influence Ca++-stimulated Cl− secretion (carbachol, ionomycin). Such results indicate that hypoxia-induced responses are specific for cyclic nucleotide pathways of electrogenic chloride secretion.

Partial reversibility by reoxygenation.

To determine the reversibility of the inhibitory effects of hypoxia on cAMP-stimulated chloride secretion, we reoxygenated cells before measurement of forskolin-stimulated ion transport. Such conditions of reoxygenation (up to 4 h) were not toxic to epithelia (determined by measurement of transepithelial resistance, data not shown). T84 cells exposed to hypoxia alone for 24 h had diminished ability to secrete Cl− (1 μM forskolin stimulation, ΔSSC 25.1 ± 0.5% of normoxic control, P < .001). As shown in figure3, monolayers reoxygenated in fresh media for time periods of 30 to 240 min rapidly and maximally recovered to 57.8 ± 2.2% of normoxic controls by 240 min (P < .001;n = 4), which indicates that such hypoxia-elicited diminutions are at least partially reversible with reoxygenation.

Reoxygenation of previously hypoxic epithelia results in partial recovery of ion transport response to forskolin. T84 intestinal epithelia were exposed to normoxia (21% O2) or hypoxia (1% O2) for 24 h, reoxygenated for the indicated periods of time (0–240 min) and examined for forskolin (10 μM)-stimulated Cl− secretion (measured as SSC). Data are pooled from four monolayers from duplicate experiments each, and results are expressed as the mean ± S.E.M.

Epithelial exposure to cobalt mimicks hypoxia. A) T84 intestinal epithelia were exposed to the indicated concentrations of CoCl2 or FeCl2 for 8 h and examined for forskolin (1 μM)-stimulated Cl−secretion. Neither CoCl2 nor FeCl2significantly influenced base-line epithelial barrier (measured as TER, inset). B) Agonist specificity for CoCl2 (250 μM) using the cAMP agonist forskolin (Fsk), the cGMP agonist E. coliheat-stable toxin and the calcium agonist carbachol (Cch) at the indicated concentrations. * Significantly different from no CoCl2 controls (P < .05). Data are pooled from 6 to 8 monolayers each, and results are expressed as the mean ± S.E.M. ΔSSC. C) cGMP generation in a cell-free assay system. Purified plasma membranes were incubated in the absence (CTL) or presence of STa (100 ng/ml) with the addition of CoCl2 or FeCl2 (333 μM). Data are pooled from four separate experiments, and results are expressed as the mean ± S.E.M. cGMP and normalized to protein concentration.

Finally, because CoCl2 but not FeCl2 mimicked hypoxia in intact epithelia, we determined whether CoCl2would inhibit stimulated GC activity in a cell-free assay. As shown in figure 5C, STa-stimulated cGMP from purified plasma membrane preparations were inhibited significantly by the addition of CoCl2 (P < .025) but not FeCl2, at concentrations shown to inhibit in intact epithelia (333 μM). Addition of FeCl2 alone significantly elevated cGMP generation (51% increase over control), although not to the level of FeCl2 and STa (84% increase over control). Notably, no elevation in cGMP was observable over baseline using the soluble GC agonist sodium nitroprusside (cGMP of 250 ± 29 vs.184 ± 13 fmol/μg cytosolic protein for control vs. 3 mM sodium nitroprusside, respectively; n = 3, P = not significant). These results, which are consistent with others (Currie et al., 1992), indicate that the particulate form of GC is predominant in T84 cells. Taken together, these data indicate that decreased electrogenic Cl− secretion elicited by hypoxia can be mimicked in normoxic condition by the metal ions that bind to heme proteins, a result that suggests a role for heme moieties as epithelial oxygen sensors.

Discussion

The studies outlined here complement a growing literature regarding tight regulation of cellular responses during conditions of hypoxia. These studies highlight the important relationship between hypoxia and intracellular levels of cyclic nucleotide second messengers and reveal that coordinated endpoint function (electrogenic Cl− secretion) can be used as a marker of a hypoxia-elicited phenotype. Two novel observations are noteworthy. First, studies using cobalt to mimic hypoxia revealed that a heme moiety may serve as an epithelial oxygen sensor. Second, at the level of the epithelium, biochemical cross-talk pathways between the second messengers cAMP and cGMP are important during such responses.

Mucosal surfaces are lined by a monolayer of epithelia that provides tissue barrier and vectorial ion transport function (Powell, 1981;Powell, 1987). Although epithelia are exposed to hypoxia in a number of disease states, only limited information is available about the direct impact of hypoxia on epithelial function. Previous studies demonstrated that renal tubule epithelia are, when compared with endothelia, quite sensitive to hypoxia and are rapidly and reversibly damaged (Tretyakov and Farber, 1995; Zimmerman et al., 1991). Furthermore, we have recently demonstrated that exposure of intestinal epithelia (T84 cells) to hypoxia modulates neutrophil-epithelial interactions and induces production and basolateral release of the proinflammatory cytokine interleukin-8 (Colgan et al., 1996). Thus our present results of decreased epithelial ion transport in response to hypoxia may serve as a mechanism of dampening fluid loss (the endpoint function of electrogenic Cl− secretion) (Powell, 1987) during periods of mucosal hypoxia.

A number of previous studies, exemplified by original work with erythropoietin (Beru et al., 1986; Goldberg et al., 1988; Schuster et al., 1989; Tsuchiya et al., 1993), have demonstrated a direct role for heme proteins in “sensing” extracellular oxygen concentrations. Such studies were substantially aided by the observation that a hypoxia-elicited phenotype can be mimicked in normoxia using cobalt, nickel or manganese, but not using iron (Goldberg et al., 1988). Although the exact mechanism of cobalt action on heme proteins has not been elucidated, a proposed model suggests that cobalt substitutes for ferrous ion within the porphyrin ring and locks heme into a deoxy state (Goldberg et al., 1988). Given that: a) both hypoxia and metal ions specifically attenuate cyclic nucleotide-stimulated (but not Ca++-stimulated) Cl− secretion (fig. 5; table1), b) in the absence of significant decreases in intracellular ATP, hypoxia decreases cAMP and cGMP (fig. 4) and c) exogenous addition of cGMP partially reverses the hypoxia phenotype (fig. 6), a potential target for cobalt and hypoxia in epithelia is heme moieties associated with cyclic nucleotide signal transduction pathways, such as GC. On the basis of our observation that reoxygenation results in a rapid (∼30 min), albeit partial, reversal of the hypoxia-mediated effect, it is unlikely that a significant reduction in enzyme level is responsible for the attenuation we observed in chloride secretion. Matthewset al. have recently demonstrated that conditions consistent with chemical hypoxia—namely, the use of metabolic inhibitors—resulted in extracellular loss of adenosine and, ironically, stimulation of electrogenic Cl− secretion (Matthews et al., 1995). We have not observed the generation of spontaneous currents in epithelia exposed to hypoxia. Carryover experiments of conditioned media derived from hypoxic epithelia to normoxic monolayers have not consistently resulted in generation of a Cl− secretory response (data not shown). Discrepancies between these results are probably explained by the substantial differences in the models. Hypoxia, as defined in our system, diminishes O2 from an ambient environment with normal glucose levels and only gradually depletes intracellular ATP (>24 h, see “Results”). The Matthews et al. model disrupted electron transport in low-glucose conditions and achieved depletion of cellular ATP levels by greater than 90% within 30 min. Thus it is possible that low levels of biologically undetectable adenosine are released over a longer period of time in our model. We have not directly addressed this issue.

The present results do not reveal the source of heme responsible for epithelial oxygen sensing. Evidence is provided that the heme moiety of GC and/or other heme molecules within cyclic nucleotide signal transduction may provide oxygen-sensing qualities. This issue is complicated by the fact that multiple forms of GC exist, including soluble GC, particulate GC and intestinal GC (Currie et al., 1992; Schulz et al., 1990). In a result consistent with previous reports (Currie et al., 1992), minimal soluble GC was observed in T84 cells. Although the various forms of GC are heme proteins, it is not known whether the active enzyme directly binds oxygen. Some evidence indicates that the heme of isolated bovine soluble GC, in fact, has the unique feature of not binding oxygen (Stone and Marletta, 1994). Additionally, Waldman et al.have demonstrated that specific porphyrins can differentially activate particulate and soluble GC (Waldman et al., 1984). With regard to our data, because hypoxia/cobalt inhibited Cl−secretory responses to STa in intact cells (fig. 5; table 1), and because STa-stimulated cGMP from purified plasma membranes was inhibited by cobalt (fig. 5), it is likely that hypoxia influences at least the plasma membrane fraction of GC.

The present work lends insight into potential biochemical cross-talk pathways between cGMP and cAMP with regard to epithelial ion transport. Notably, hypoxia-elicited decreases in ion transport were specific for cyclic nucleotide- (cAMP and cGMP) but not calcium-stimulated ion transport (table 1), a result that suggests some degree of similarity in signaling. The observed decreases in forskolin-stimulated ion transport after specific inhibition of GC (fig. 6) directly imply significant cAMP/cGMP cross-talk at the level of epithelial ion transport, and direct elevation of intracellular cGMP in hypoxic cells normalized cAMP-stimulated responses. Unlike other cell systems, cGMP and cAMP signaling in epithelia do not appear to be antagonistic (Barrett, 1993), and in fact, some evidence suggests that shared pathways exist. Our data indicate that hypoxia inhibits cAMP signal transduction at levels proximal (adenylate cyclase) and distal (inhibition of cAMP analog response; see table 1) to cAMP generation. Also, because this inhibitory effect is at least in part dependent on decreased cellular cGMP and is mimicked by cobalt, our results indicate a multifaceted epithelial response to hypoxia involving inhibition of regulatory heme proteins. A potential site for inhibition of cAMP responses dependent on cGMP includes protein kinase A, which is cross-activated by cGMP. Another potential target includes specific cAMP phosphodiesterases, which have been shown to be negatively regulated by cGMP (Acker, 1994). Evidence for such a pathway includes studies demonstrating that T84 cells do not possess specific cGMP-dependent protein kinases, so it is likely that responses to STa (i.e., elevation of cGMP) are mediated by cross-activation of protein kinase A (Forte et al., 1992); this would explain the lack of response to cGMP analogs alone and the enhanced responses to forskolin in both hypoxic and normoxic epithelia. Finally, a third candidate for such regulation is the cystic fibrosis transmembrane regulator (CFTR), which is regulated by both cAMP and cGMP pathways during electrogenic Cl− secretion (Barrett, 1993). T84 cells, used in these experiments, have a well-defined CFTR (Gregoryet al., 1990). These findings indicate that pathophysiologically relevant conditions such as hypoxia may directly influence intracellular signaling events, resulting in altered endpoint functional responses such as ion transport.

Acknowledgments

The authors gratefully acknowledge the superb technical assistance of Ms. Andrea Dzus. This work was supported by National Institutes of Health research grant DK50189 to S.P.C.